WO2020171764A1

WO2020171764A1 - Cryo-em specimen preparation

Info

Publication number: WO2020171764A1
Application number: PCT/SE2020/050191
Authority: WO
Inventors: Xiaodong Zou; Jingjing Zhao; Hongyi Xu
Original assignee: Xiaodong Zou; Jingjing Zhao; Hongyi Xu
Priority date: 2019-02-21
Filing date: 2020-02-19
Publication date: 2020-08-27

Abstract

The present disclosure relates to a method for cryo-electron microscopy (cryo-EM) specimen preparation. The method comprises the steps: a) providing an EM sample grid; b) applying a sample dispersed in a liquid onto a sample face of the EM sample grid; c) providing a pressure gradient through the EM sample grid in order to force a portion of the liquid through the EM sample grid and remove the portion of liquid; and d) immersing the EM sample grid into a cryogenic bath. The disclosure further relates to auxiliary suction modulesand cryo-EM specimenpreparation apparatus for performing such a method.

Description

Cryo-EM Specimen Preparation

TECHNICAL FIELD

The present invention relates to a method for preparing specimens for cryo-EM, as well as a cryo-EM specimen preparation apparatus and an auxiliary suction module for performing such a method.

BACKGROUND ART

Knowing the atomic structure of biomolecules is essential for understanding their functions. Cryo-electron microscopy (cryo-EM), including single particle imaging, microcrystal electron diffraction (MicroED) and electron cryotomography (CryoET), is an important technique for studying such biomolecules, either as isolated single particles, organelles or as microcrystals. Because cryo-EM studies are carried out in vacuum, the biomolecules/crystals will collapse if unprotected. An increasing widely used means of providing such protection is to rapidly cool the biomolecules in solvent, which leads to the formation of vitrified (non-crystalline) ice around the molecules/microcrystals. Such methods are termed cryo-electron microscopy (cryo-EM) methods, and the Nobel Prize in Chemistry was awarded in 2017 for the

development of cryo-EM.

Specimen preparation for cryo-EM typically comprises the following steps: deposition of a sample droplet on an EM sample grid, formation of a thin film by removal of excess liquid, and vitrification by immersion in a cryogen, such as liquid ethane. The removal of excess liquid is typically performed using blotting paper, although other methods such as sucking up a portion of the sample droplet using a capillary are also known. A review of cryo-EM specimen preparation methods is provided in Arnold et al. "Miniaturizing EM Sample Preparation:

Opportunities, Challenges, and "Visual Proteomics"", Proteomics, 18, 1700176.

The specimen preparation method above may be performed by skilled technicians. However, a number of cryo-EM specimen preparation apparatus are commercially available in order to partially automate and increase reproducibility of the specimen preparation process. The term "controlled environment vitrification system" (CEVS) is commonly used for a cryo-EM specimen preparation apparatus. Such apparatus typically comprise a temperature- and humidity-controlled specimen preparation chamber, a plunge rod, one or more blotting arms, and a cryogenic bath. The plunge rod is initially located in the chamber and is arranged to hold a tweezer clamping a EM sample grid. A side port in the chamber allows a micropipette to be inserted into the chamber in order to deposit a sample droplet on the EM sample grid. The blotting arms are arranged in the chamber and controllably blot the EM sample grid after sample deposition. After blotting, the plunge rod plunges the EM sample grid downwards into the cryogenic bath. The tweezer clamping a EM sample grid may then be removed from the plunge rod and the EM sample grid is placed in cryogenic storage for transport to the electron microscope.

Devices utilizing other methods for specimen preparation, such as devices based on inkjet printing techniques for sample deposition onto the EM sample grid, are known.

There remains a need for improved means for preparing specimens for cryo-electron microscopy (cryo-EM).

SUMMARY OF THE INVENTION

The inventors of the present invention have identified a number of shortcomings with prior art means of preparing a specimen for cryo-electron microscopy (cryo-EM). Conventional blotting methods remove a large proportion of the sample, often in excess of 99.9%, meaning that the sample droplet must have a high sample concentration and that potentially extremely valuable sample is wasted. Blotting also exposes the sample to shear forces and contaminants which can damage or deteriorate the quality of the sample. Moreover, the blotting methods are often difficult to reproduce and may work poorly for samples dispersed in viscous liquids such as PEG solutions or lipid cubic phase.

Inkjet printing based methods potentially circumvent some of these problems, but they have an extremely high cost and are complicated to use. Although inkjet-based methods deposit only a few pico- to nanoliters of sample, they must be primed with about 1 microliter of sample, and the sample concentration required is similar to that of conventional blotting methods. Moreover, it is unsure if inkjet-based methods can handle viscous sample liquids, or if the print heads can handle or tolerate microcrystalline samples. This may limit the utility of inkjet-based methods for MicroED specimen preparation.

It would be advantageous to achieve a means for cryo-EM specimen preparation that overcomes, or at least alleviates, some of the above mentioned shortcomings. In particular, it would be desirable to enable a means for cryo-EM specimen preparation that requires lower sample concentrations and wastes less sample.

To better address one or more of these concerns, a method for cryo-EM specimen preparation is provided, having the features defined in the appended independent claim.

The method for cryo-electron microscopy (cryo-EM) specimen preparation comprises the following steps: a) providing an EM sample grid; b) applying a sample dispersed in a liquid onto at least a first face of the EM sample grid; c) providing a pressure gradient through the EM sample grid in order to force a portion of the liquid through the EM sample grid and remove the portion of liquid; and d) immersing the EM sample grid into a cryogenic bath.

By providing a pressure gradient through the EM sample grid, a portion of the liquid is forced through the EM sample grid. At the same time, the portion of the liquid is removed by the pressure gradient, so that a suitably thin film of sample dispersed in liquid is formed without excessive removal of sample materials. Consequently, sample solutions having a magnitude lower concentration may be used in preparation of the specimen. Since it is no longer necessary to use blotting papers that is in contact with the sample, the sample is not exposed to contaminants from the blotting papers and nor is it exposed to the shear forces generated by the blotting process. By carefully controlling the conditions used for providing the pressure gradient, the method is highly reproducible, and excellent sample films may be consistently obtained. Moreover, the method is simple, robust and relatively cheap and easy to

implement. It can also be adapted to existing cryo-EM vitrification devices. Step c) of the method, i.e. providing a pressure gradient through the EM sample grid, may be performed by applying a suction to a second face of the EM sample grid, the second face being the opposite face to the first face. Whenever sample is applied only to a single face of the EM sample grid, the first face may alternatively be termed herein the sample face, whereas the second face may be referred to as the rear face of the EM sample grid. Suction-generating apparatus are widely available, making this method cheap and easily applicable. Moreover, since the only contact with the EM sample grid is on the rear side, there is little risk of accidently removing or contaminating the specimen.

Performing step c) by applying a suction to the rear/second face of the EM sample grid may be performed in any one of a number of manners.

The EM sample grid may be clamped in an essentially vertical position by a tweezer, and the suction may then be applied by a suction nozzle brought into in adjacency or contact with the second face of the EM sample grid. This method is readily applicable in combination with existing cryo-EM specimen preparation apparatus, since such apparatus typically clamp the EM sample grid using a tweezer, and include side ports allowing access to the rear of the EM sample grid when the sample grid is clamped in such a position.

The suction nozzle may arranged to move in a scanning motion over the second face of the EM sample grid. For example, the suction module may be moved in a one or more pre

programmed patterns over the second face. This allows for a controlled variation of specimen preparation conditions across the EM sample grid.

The suction nozzle may be covered totally, or covered partially, or not covered with a porous support. This provides support for the EM sample grid and may help prevent bending or distortion of the grid.

The suction nozzle may comprise a plurality of nozzle sub-orifices, each nozzle sub-orifice being arranged to provide an individual flow direction and/or flow rate through the nozzle sub-orifice. This allows for a controlled variation of specimen preparation conditions across the EM sample grid.

A suction nozzle may be brought into contact with the second face of the EM sample grid, and this suction may be utilized in a step of lifting and/or transferring the EM sample grid by using the suction applied by the suction nozzle. For example, the grid may be lifted and the first face of the grid may be placed in contact with a sample droplet using suction provided at the second face of the EM sample grid. This provides a suitably thin film of sample on the EM sample grid in a single combined step (i.e. combining steps b) and c) of the method above). Alternatively, or in addition, the EM sample grid with applied sample may be transferred to a position directly above a cryogenic bath using suction provided at the second face of the EM sample grid in order to carry the grid. By then removing suction, the EM sample grid may fall into the cryogenic bath, thus performing step d) of the method above. Thus, by utilizing the suction in a step of lifting and/or transferring the EM sample grid, the inventive method may be consolidated and fewer components may be required to implement the method.

The second face of the EM sample grid may be rested upon a porous support. In such a case, suction may be applied through the porous support. This allows specimen preparation to be performed using cheap and widely available laboratory apparatus such as a Buchner or Hirsch funnel, a Buchner flask, and a source of suction such as a vacuum pump, evaporator or water aspirator. The porous support may assist the removal of excess liquids, making the method especially advantageous for samples in viscous liquid, as described below.

The step c) of providing a pressure gradient through the EM sample grid may alternatively be performed by applying a positive pressure to the first face of the EM sample grid, i.e. blowing the liquid through the sample grid.

The liquid that the sample is dispersed in may be a viscous liquid, such as an aqueous PEG solution or lipid cubic phase. Many proteins, for example membrane proteins, require crystallisation from a viscous mother liquid, and such specimens are difficult to be reliably prepared using conventional blotting methods. The method provides a reliable means of preparing such specimens from viscous liquids. Viscous liquids may for example be liquids having a viscosity of 10 mPa.s or greater when measured at 25°C and 1 atm pressure.

According to another aspect of the invention, an auxiliary suction module for a cryo-EM specimen preparation apparatus is provided, as defined in the independent claims. The auxiliary suction module is adapted to perform at least step c) of the method described herein, but may also be adapted to perform steps b) and d). According to one envisaged implementation of the auxiliary suction module, the auxiliary suction module comprises a mechanical arm. The mechanical arm comprises a suction nozzle arranged to be connected to a source of partial vacuum. The mechanical arm is arranged to controllably provide translational motion of the suction nozzle, such that the suction nozzle may be brought into adjacency or contact with a second face of an EM sample grid. However, other alternative implementations of auxiliary suction modules adapted to perform step c) and optionally steps b) and d) of the method described herein are readily conceivable to the skilled person.

Such auxiliary suction modules may be provided as an aftermarket accessory for existing commercial cryo-EM specimen preparation apparatus. Thus, using the auxiliary suction module, pre-existing cryo-EM specimen preparation apparatus may be provided with the capability of preparing cryo-EM specimens as described herein. The auxiliary suction module may be a free-standing module arranged such that the mechanical arm can extend into a specimen preparation chamber of the cryo-EM specimen preparation apparatus, e.g. through a side port in the specimen preparation chamber. Alternatively, the auxiliary suction module may be adapted to be mounted to a side port of a specimen preparation chamber, or may be adapted to be mounted fully or partially within a specimen preparation chamber. For example, the auxiliary suction module may be adapted to replace one or more blotting arms of the cryo- EM specimen preparation apparatus.

According to a further aspect of the invention, a cryo-EM specimen preparation apparatus is provided, as defined in the independent claims. The cryo-EM specimen preparation apparatus is adapted to perform at least step c) of the method described herein, but may also be adapted to perform steps b) and d).

According to one envisaged implementation of the cryo-EM specimen preparation apparatus, the apparatus comprises a retractable plunge rod and a volume arranged to accommodate a cryogenic bath. The retractable plunge rod is adapted to directly or indirectly hold an EM sample grid and to plunge the EM sample grid into the bath volume (i.e. plunge the EM sample grid downwards into a cryogenic bath when the specimen preparation apparatus is arranged in its normal orientation and is equipped for operation). The apparatus comprises a

mechanical arm. The mechanical arm comprises a suction nozzle arranged to be connected to a source of partial vacuum. The mechanical arm is arranged to controllably provide translational motions of the suction nozzle, such that the suction nozzle may be brought into adjacency or contact with a second face of an EM sample grid. Such a cryo-EM specimen preparation apparatus may perform the method for cryo-EM specimen preparation as described herein.

The mechanical arm may be arranged to move the suction nozzle in three translational dimensions. The mechanical arm may be arranged to only move the suction nozzle in a plane, i.e. in two translational dimensions, for example by a rotational movement, or to controllably extend and retract the suction nozzle in a single translational dimension. Thus, the mechanics and control system of the cryo-EM specimen preparation apparatus may be simplified since motion of the arm is not necessarily required in three dimensions.

The retractable plunge rod may be a separate component from the mechanical arm, and may be adapted to hold a tweezer clamping an EM sample grid and to controllably plunge the tweezer clamping the EM sample grid into the bath volume. Alternatively, the retractable plunge rod may comprise the mechanical arm, i.e. the retractable plunge rod and mechanical arm may be essentially the same component. This single component may be capable of controllably performing the steps of holding, transferring and plunging the EM sample grid as described herein, and may further be capable of applying the sample to the EM sample grid as described herein.

However, other alternative implementations of auxiliary suction modules adapted to perform step c) and optionally steps b) and d) of the method described herein are readily conceivable to the skilled person.

Further objects, advantages and novel features of the present invention will become apparent to one skilled in the art from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the present invention and further objects and advantages of it, the detailed description set out below should be read together with the accompanying drawings, in which the same reference notations denote similar items in the various diagrams, and in which:

Fig. 1 is a flowchart schematically illustrating the method of the present invention;

Fig. 2a schematically illustrates the "horizontal" method and apparatus of the present invention;

Fig. 2b schematically illustrates the "vertical" method and apparatus of the present invention;

Fig. 2c schematically illustrates a further variation on the method and apparatus of the present invention;

Fig. 3 schematically illustrates an auxiliary suction module mounted to a cryo-EM

specimen preparation apparatus;

Fig. 4 schematically illustrates a cryo-EM specimen preparation apparatus according to the present invention;

Fig. 5 presents a series of TEM images for single particle specimens of varying

concentrations prepared by the inventive method (a-b) and conventional method (c);

Fig. 6 presents a series of TEM images for crystal specimens prepared by conventional method (a-b) and the inventive methods (c-d);

Fig. 7 presents a series of TEM images and MicroED diffraction patterns for crystal specimens prepared under different conditions using the inventive method;

Fig. 8 presents a series of TEM images and MicroED diffraction patterns for crystal specimens prepared in viscous solutions by conventional method (a-c) and the inventive method (d-f);

Fig. 9 presents a series of TEM images for GroEL specimens prepared on a grid

comprising a continuous ultrathin carbon film; and Fig. 10 presents a series of TEM images for cryoET specimens prepared by the inventive method (c-d) and conventional method (a-b).

DETAILED DESCRIPTION

Since specimen preparation is one of the main bottlenecks in cryo-EM, new specimen preparation methods need to be developed to address problems of sample consumption, grid reproducibility, viscous mother liquid, etc. By applying a pressure difference through the EM sample grid, the inventors of the present invention have developed a simple and cheap method to prepare specimens for cryo-EM (called Preassist). This method is applicable to all samples studied with different cryo-EM techniques, including but not limited to single-particle cryo-EM, electron cryotomography (cryoET) and microcrystal electron diffraction (MicroED). The method can reduce more than 20 times the amount of sample required for single-particle cryoEM experiments as compared to the conventional deposit-blot-plunge method. The method can also be used to prepare cryo-EM specimens of protein crystals grown in viscous mother liquid for MicroED experiments, which the conventional method often fail.

Method

A flowchart illustrating the inventive method is provided in Figure 1. The method comprises the steps of: a) providing an EM sample grid (with or without glow-discharging); b) applying a drop of the sample dispersed in a liquid onto one face or both faces of the EM sample grid; c) providing a pressure gradient through the EM sample grid in order to force a portion of the liquid through the EM sample grid and remove the portion of liquid; and d) immersing the EM sample grid into a cryogenic bath. In step a) an EM sample grid is provided. The EM sample grid used may be any TEM sample grid known in the art. Sample grids are typically circular and have a standard diameter of 3.05 mm. The grids are typically of a metal or metal alloy, such as copper, nickel, molybdenum, rhodium, titanium, gold, tungsten, steel, or alloys thereof, but may also be of a structural polymer such as nylon. The number and diameter of holes in the grid is defined by the mesh size, and commercially available grids typically range from 50 mesh to 600 mesh. The grid is typically coated with a thin electron-transparent film in order to support the sample on the grid. The thin film may be holey or lacey, and is typically made of carbon, although thin films made of gold or thermoplastic resin such as formvar are also known. Continuous thin films, such as ultra-thin carbon film, graphene film or bio-based films may be used. Continuous films may assist in distributing the applied pressure gradient more evenly across the grid, and thereby provide more homogenous ice thickness across the grid. The thin film may be a Quantifoil^® film, i.e. a thin continuous or perforated carbon film having a well-specified hole size, multiplicity, shape and arrangement. The holes in the film may preferably be circular, although "multi-patterned"-holed film may also be used. Film hole sizes may range from about 0.6 pm to about 8 pm, preferably from about 1.2 pm to about 3.5 pm. Depending on the size, multiplicity, shape and arrangement of the holes, the pressure gradient applied through the grid may have to be adapted to achieve optimal results. Prior to use, the EM sample grid may be treated, for example by glow discharge or plasma etching, in order to increase the hydrophilicity of the grid and improve aqueous sample spreading.

In step b) a sample dispersed in a liquid is applied onto one face or both faces of the EM sample grid. The sample may be any sample that may be subjected to cryo-electron microscopy, including but not limited to macromolecules, such as biological macromolecules (e.g. proteins), and small molecules. The sample may be single particles or may be crystals in the form of e.g. microcrystals. The sample is dispersed in a liquid. The liquid may typically be an aqueous liquid, such as an aqueous solution of a salt or polyalkylene glycol, or a lipid-water system (e.g. lipid cubic phase). One or more drops of the sample dispersed in liquid may be applied to one face of the EM sample grid (i.e. the side of the grid covered by the holey thin film) or both faces of the grid using techniques known in the art, such as micro-pipetting. The sample droplet(s) may be any appropriate volume, commonly about 3 pi. In step c) a pressure gradient is provided through the EM sample grid in order to force a portion of the liquid through the EM sample grid and remove a portion of the liquid. By "force a portion of the liquid through the EM sample grid", it is meant to compel the liquid through the EM sample grid by means of the applied pressure gradient. This may encompass either pushing the liquid through the grid using a positive pressure applied at the sample/first face of the EM sample grid, or pulling the liquid through the grid by using a negative pressure

(suction) applied at a rear/second face of the EM sample grid. The pressure gradient may be provided simultaneously with or successive to applying the sample droplet. Whether the pressure gradient is applied simultaneously with or successively to applying the sample droplet depends on from which side of the grid the pressure gradient is applied (i.e. a positive pressure applied at the sample face would impede deposition of the sample droplet and therefore must be performed after droplet deposition), as well as on other practical considerations.

The pressure gradient may be applied using any means known in the art. For example, if a positive pressure is to be applied, a pump, compressor or source of compressed gas such as a gas canister may be used. If a negative pressure (suction) is to be applied, applicable sources include but are not limited to any suitable suction pump, such as a vacuum pump, water aspirator or membrane pump of a laboratory evaporator. The exact pressure gradient and time period required in order to obtain an appropriate sample film thickness depend on a number of factors including the viscosity of the liquid, the size, number and arrangement of holes in the grid thin film. Adapting the pressure gradient to obtain an appropriate sample film thickness is well within the capabilities of the skilled person.

If the pressure gradient through the EM sample grid is obtained by applying suction to the rear face of the EM sample grid, this may be done in any one of a number of manners.

The simplest means of applying suction to the rear/second face of the EM sample grid is by using classical laboratory equipment, as schematically illustrated in Figure 2a. This is termed herein the horizontal method, defined with respect to the orientation of the grid during suction. A Buchner flask 201 is connected to a suction pump 203, such as a water aspirator. A pressure gauge 205 may be arranged between the flask and the suction source in order to assist in the reproducibility of the specimen preparation. A porous support 207 is placed upon the Buchner flask 201, such as a filter paper (as illustrated) or Buchner funnel (with filter paper or sintered glass frit). The grid 209 is placed on the porous support 207 and suction is applied by suction pump 203 through the Buchner flask 201. A sample droplet is applied to the grid 209 and suction is applied for a desired period in order to provide a thin sample film. Finally, the grid is lifted using tweezers (not shown) and is plunged into a cryogenic bath, such as a liquid ethane bath. Note that it is not strictly necessary that suction is applied prior to sample deposition, but that establishing a reliable suction level prior to sample deposition may improve ease of control and method reproducibility.

An alternative means of applying suction to the rear/second face of the EM sample grid is by clamping the grid 209 using a tweezer 211, and applying suction using a suction nozzle 213 brought into adjacency with the rear/second face of the EM sample grid 209, as shown in Figure 2b. This is termed herein the vertical method. It can be seen that the suction nozzle 213 is arranged in fluid communication with a suction pump 203, such as a vacuum pump, by a length of tubing 215. The EM sample grid 209 is held in an essentially vertical position and a sample droplet is deposited on the sample face of the grid. The suction nozzle 213 with suction provided is then brought into adjacency or contact with the rear face of the sample grid 209 sufficient to cause liquid to pass through the grid and provide a thin film of sample. The EM sample grid 209 is then immersed into a cryogenic bath in the conventional manner.

This method is compatible with currently available controlled environment vitrification systems (CEVS), since these systems typically comprise a port allowing access to the rear/second face of the sample grid when suspended in the apparatus, either directly, or by turning the grid using the plunge arm such that the rear side faces a port arranged for sample deposition.

If the vertical method is used, the suction nozzle 213 may be moved in a scanning motion over the rear face of the EM sample grid 209. The scanning motion may be pre-programmed, or may be controlled dynamically by a user. This allows precise control of the sample preparation conditions at a variety of points across the sample grid, for example allowing multiple preparation conditions across a single grid. The scanning motion may for example be combined with controlled variation in the strength or duration of suction provided at each point in the grid. Alternatively, or in addition, the suction nozzle may be used to lift and/or transfer the EM sample grid using the suction applied by the suction nozzle. This is illustrated in Figure 2c. It can be seen that the EM sample grid 209 is initially arranged with the sample face resting against a surface 217. The suction nozzle 213 is applied to the rear face of the sample grid 209 and used to lift and transport the grid to a sample droplet 219 provided on a substrate 221, such as a glass laboratory slide. A porous support can be positioned between sample grid 209 and suction nozzle 213 to assist the lifting of the EM sample grid, as well as assist liquid removal. The sample face is brought into contact with the sample droplet 219, thereby promptly providing a thin sample film on the sample face of the grid 209. The suction nozzle 213 is then used to transport the sample-coated grid 209 to a position directly above a cryogenic bath 223. By breaking the suction to the suction nozzle 213, the force attaching the grid 209 to the nozzle 213 is removed and the grid 209 falls into the cryogenic bath 223, where it is immersed. It can be appreciated that such a method significantly reduces the time required for specimen preparation and is amenable to high-throughput specimen preparation, especially if a plurality of suction nozzles are used. Thus, the method may be used for kinetic studies, by sampling a reaction mixture at regular intervals and then immediately preparing the samples for cryo-EM analysis.

In step d) the EM sample grid is immersed into a cryogenic bath. Liquid ethane baths having a temperature of approximately -172 °C are typically used as the cryogen. The ethane bath is held in the liquid state by immersion in a liquid nitrogen bath, having a temperature of approximately -196 °C. The immersion may be performed by actively plunging a fixed (e.g. tweezer-clamped) sample grid into the cryogen, or by allowing the grid to fall into the cryogen. After immersion in the cryogen, the prepared EM sample grid may be retrieved from the cryogenic bath and transferred to the electron microscope using conventional means known in the art.

Auxiliary suction module

An auxiliary suction module may be provided for assisting in performing the method as described herein. The auxiliary suction module is intended as an aftermarket accessory to provide existing commercially available CEVS with the functionality required for performing at least step c) of the method as described, since commercially available machines in combination with skilled operators are already equipped to perform the remaining steps of the method. There are many conceivable means of implementing such a module, but an exemplifying implementation is provided in Figure 3. Figure 3 schematically illustrates an auxiliary suction module 200, mounted to a cryo-EM specimen preparation apparatus 300. The cryo-EM specimen preparation apparatus 300 comprises a specimen preparation chamber 301, a retractable plunge rod 303 adapted to hold a tweezer 211 clamping an EM sample grid 209, and a bath volume 305 arranged to accommodate a cryogenic bath 223. The auxiliary suction module 200 is fixed to a side port of the specimen preparation chamber 301 at the rear side of the sample EM grid. The auxiliary suction module 200 comprises a mechanical arm 250. The mechanical arm 250 comprises a suction nozzle 213 arranged to be connected to a source of partial vacuum 203 (via the mechanical arm 250 in the illustrated instance). The mechanical arm 250 is arranged to controllably provide translational motion of the suction nozzle 213, such that the suction nozzle may be brought into contact with or adjacent to the rear face of the EM sample grid, thus performing step c) of the inventive method. In the embodiment exemplified in Figure 3, the mechanical arm 250 may move only in a single translational direction forward/backwards (as illustrated by arrow 253). However, a mechanical arm capable of controlled movement in two, or all three translational dimensions is equally feasible. In the example illustrated herein, the blotting arms 307 of the cryo-EM specimen preparation apparatus, although not required for the inventive method, are allowed to remain mounted in the chamber 301, since they do not interfere with performing the method. However, the blotting arms 307 may also be dismounted if desired. Alternatively, the mechanical arm of the auxiliary suction module may replace the blotting arm(s), or consist or comprise of an appropriately modified blotting arm(s).

A cryo-EM specimen preparation apparatus

A cryo-EM specimen preparation apparatus may be provided for assisting in performing the method as described herein. There are many conceivable means of implementing such an apparatus, but an exemplifying implementation is schematically illustrated in Figure 4. The apparatus 300 comprises a specimen preparation chamber 301, a retractable plunge rod 303 and a bath volume 305 arranged to accommodate a cryogenic bath 223. The retractable plunge rod 303 is adapted to hold a tweezer 211 clamping an EM sample grid 209 and to controllably plunge the grid 209 downwards into the cryogenic bath 223. The apparatus 300 further comprises a mechanical arm 250 arranged inside of the specimen preparation chamber 301, at the rear side of the EM sample grid. The mechanical arm 250 comprises a suction nozzle 213 arranged to be connected to a source of partial vacuum 203. The mechanical arm 250 is arranged to controllably provide translational motion of the suction nozzle 213 (as indicated by dotted line 214), such that the suction nozzle 213 may be brought into contact with or adjacent to the rear face of the EM sample grid 209. This is illustrated in this example as being achieved by a rotational motion of the mechanical arm 250, although it can also be achieved by other means, such as using an arm designed for reciprocal motion as illustrated in Figure 3 above.

Suction nozzle

The following features relate to any of the methods, modules or apparatus as defined herein that utilize a suction nozzle. The suction nozzle may comprise a single orifice, a plurality of nozzle sub-orifices, each nozzle sub-orifice being arranged to provide an individual fluid flow direction and/or fluid flow rate through the nozzle sub-orifice. This allows for a controlled variation of specimen preparation conditions across the EM sample grid. The suction nozzle may have a total nozzle orifice diameter that is smaller than the diameter of an EM sample grid, as this assists in preventing the grid being sucked into the nozzle. Alternatively, the suction nozzle may have a total nozzle orifice diameter that is greater than, or equal to a diameter of an EM sample grid. EM sample grids typically are circular and have a diameter of 3.05 mm. The suction nozzle may be essentially circular and/or may have a total nozzle orifice diameter of about 1 mm or greater, such as about 2 mm or greater, such as about 3 mm or greater, such as about 4 mm or greater. By total orifice diameter it is meant the smallest diameter of the entire nozzle orifice, regardless of whether the nozzle orifice comprises a single orifice or a plurality of sub-orifices. Use of a suitably large orifice diameter reduces the requirement for excessive precise positioning of the suction nozzle. By utilizing an orifice diameter greater or equal to the size of the EM sample grid, it is ensured that suction is applied across substantially the entirety of the grid, without requiring excessively precise placement of the suction nozzle or movement of the suction nozzle lateral to the grid. The suction nozzle may comprise a grid support arranged transversely across the nozzle orifice in order to support the EM sample grid when the suction is applied. This assists in preventing bending of the EM sample grid, or assists in preventing the EM sample grid from being sucked into the suction nozzle if the sample grid is not otherwise fixed or supported. The grid support may be porous, and may for example be a strip or disc of filter paper or sintered glass.

The invention will now be described in more detail with reference to certain exemplifying embodiments and the drawings. However, the invention is not limited to the exemplifying embodiments discussed herein and/or shown in the drawings, but may be varied within the scope of the appended claims. Furthermore, the drawings shall not be considered drawn to scale as some features may be exaggerated in order to more clearly illustrate certain features.

Examples

There are many parameters that may be adapted to adjust the vitrified ice thickness.

However, we recommend standardizing all hardware, such as tube size and pumping machine type in order to increase reproducibility. Different protein samples may have different behaviours. Based on the samples, we may need to change the pumping speed/pressure gradient, quantifoil hole size, as well as the sucking-plunging time in order to optimize the prepared specimens. For protein crystals that are grown at room temperature and are relatively stable, the horizontal method provides good results at a low cost. For samples in some very viscous liquids, it is strongly recommend to use the horizontal method. However, for single particle samples or some sensitive protein crystals, it's better to use the vertical method which can be implemented using existing cryo-EM vitrification devices, such as the FEI Vitrobot, Leica EM GP and Gatan Cryoplunge systems, since these devices can provide temperature and humidity control.

Equipment

Common equipment

Micropipettor and tips, 0.5-10 pi (Eppendorf Research plus variable micropipettor,

#3116000015, Eppendorf tips 0.1-10 mI or any other similar products)

Tweezers (Dumont Tweezer, style5 #72705-01, or any other similar products)

Filter paper (Munktell #110067, or other similar products)

Quantifoil with circular holes (R 1.2/1.3, R2.0/1, R3.5/1) Glow-discharge apparatus (PELCO easiGlow™ 9100)

Cryo grid box (CGB4-1 SWISSCI Cryo grid box or others)

FEI coolant container Ethane and liquid nitrogen Vacuum aspirator or pump (PC 3001 VARIO, or other similar products)

For vertical method

Glass tube (Seroloical 2 mL, or other similar products)

Plastic tubing

2-way tube junction For horizontal method

Water flow

Pressure meter (PELCO^R 2245 Mini Hot Vac, or other similar products)

Buchner flask (GLASSCO 500 mL or other similar products)

3-way T-junction Plastic tubing

Procedure 1. Vertical method

Various types and size of pumps, tubes and tubing may be used. However, it is preferable that all tubes and tubing should keep its original shape and not shrinking during the pumping process, since this may otherwise affect the pressure gradient produced on the two sides of a TEM grid. Once the apparatus is fixed, it's preferred to only adjust the quantifoil hole size, pumping speed, and/or suction time in order to control the vitrified ice thickness.

1.1 Start FEI Vitrobot (fill the humidifier, switch on, set the application parameters). Typical suitable parameters for the Vitrobot are 100% humidity, 4°C, blot total 0, wait time 0, drain time 0, check 'Use Footpedal', 'Humidifier off during process', and 'Skip Grid Transfer'. The parameters, such as humidity and temperature, can be changed if necessary.

1.2 Glow discharging the grids (e.g. current 20 mA, glow 40 s, hold 10s).

1.3 Prepare the FEI coolant container.

1.4 Use a tweezer to pick up a glow-discharged grid, mount it onto the connection grove of the Vitrobot. Place the coolant container onto the platform ring.

1.5 Completely cover, partially cover or not cover one end of the glass tube with a fastened strip of filter paper in order to provide a grid support during suction (Alternatively, the grid can be preclipped by a c-clip ring and an autogrid support). Connect the other end of the glass tube to the source of suction using plastic tubing and initiate the source of suction.

1.6 Use a micropipettor to intake 3 pi (or other volumes) of sample solution, and apply it onto one face or both faces of the grid.

1.7 In the case where no cover/support is used, move the open end of the glass tube adjacent to the rear face of the grid. In the cases where complete or partial cover/support is used, move the end of the glass tube having the grid support to the rear face of the grid until the grid touches the support (ie. filter paper). Retain for about 5 -10 s.

1.8 Withdraw the glass tube from the grid and initiate plunge freezing of the grid using the Vitrobot.

1.9 Cryotransfer the grid into a cryo-grid box.

2. Horizontal method

This method may use a vacuum (water-flow) aspirator or a vacuum pump in order to provide suction. The degree of suction (as measured at a pressure gauge arranged in the line between the source of suction and the Buchner flask), the size of flask, the size of the mouth of the flask, the time from applying sample to plunging freeze, as well as the hole size of the film on the grid will all affect the ice thickness. The total period during which applying-sucking- plunging is performed is usually adjusted to approximately 5 to 10 seconds. 2.1 Prepare the FEI coolant container.

2.2 Glow discharging the grids (same parameters as above).

2.3 Connect the Buchner flask to the source of suction and place a filter paper over the mouth of the Buchner flask (fix the filter paper if necessary to avoid the paper falling into the flask).

2.4 Use a pair of tweezers to pick up a glow discharged grid and place it in the middle of the filter paper.

2.5 Initiate suction.

2.6 Use a micropipettor to intake 3 pi (or other volumes) sample solution, apply it onto the grid, pick it up and plunge freeze it, preferably within 5 - 10 seconds (For some very viscous sample solutions, the sample volume may for example be decreased to 1 or 2 mI).

2.7 Cryotransfer the grid into a cryo-grid box.

Apoferritin specimens

In order to calibrate the amount of sample required for specimen preparation by this invention, specimen preparation for single particle cryo-EM was tested using the protein apoferritin (481.2 kDa; Sigma-Aldrich, A3641, 35 mg/mL). This protein solution was further diluted into 3.5 mg/mL, 0.35 mg/mL and 0.18 mg/mL by a filtered cryo-compatible buffer (20 mM Tris-CI, PH 7.5; 150 mM NaCI, filtered using a centricon 0.2 pm filter). In order to control the temperature and humidity, we used the vertical method implemented within a Vitrobot^® (FEI, Mark IV), as described above. The thickness of the vitrified ice can be controlled by changing the pumping speed/pressure gradient and the time period during which the pressure gradient is applied. By adjusting these two parameters, we can obtain suitable ice thickness in a quite large area. A cryo-TEM image of the specimen obtained by the inventive method using a 3.5 mg/ml concentration of apoferritin is shown in image (a) of Figure 5, and a

corresponding image of the specimen obtained by the inventive method using a 0.35 mg/ml concentration of apoferritin is shown in image (b). For comparative purposes, a specimen was also prepared by the conventional blotting method using 3.5 mg/ml apoferrin. A cryo-TEM image of this reference specimen prepared by blotting is shown as image (c) of Figure 5. Generally, using a regular Vitrobot blotting specimen preparation, the appropriate concentration of apoferritin is about 3.5 mg/ml. However, using such a concentration with the inventive method, a too-dense specimen is obtained (image (a)). However, If a concentration of 0.35 mg/ml is used for the inventive method (image (b)), the density of particles on the specimen prepared by the inventive method is still larger than the reference (image (c)), despite the sample being diluted by an order of magnitude. This demonstrates that the inventive method can decrease the sample consumption by an order of magnitude comparing to the traditional blotting method.

Preparation of lysozyme protein crystal specimens

The results were further verified by preparing a lysozyme protein crystal specimen for MicroED, as illustrated in Figure 6. In this example, the horizontal method was used for specimen preparation. Images (a) and (b) shown low magnification TEM images of specimens prepared using the conventional Vitrobot method. Images (c) and (d) show specimens prepared using the same sample concentration, but instead using the inventive method. The protein crystals are seen in the images as long, thin lines, as identified by arrow 601. Again, it can be seen that more than 10 times the amount of protein is preserved on the specimen using the inventive method as compared to the reference blotting method. Another point of note is that the inventive method provides a more uniform vitreous ice thickness, as can be seen by the more uniform brightness of the grid holes (image (c)) and Quantifoil holes (image (d)) as compared to the reference (images (a) and (b) respectively).

Tests were also performed during lysozyme specimen preparation in order to determine the optimal suction strength in relation to hole size of the Quantifoil film. The results are shown in Figure 7, as both TEM images and MicroED diffraction patterns for each specimen. The x-axis denotes hole size (increasing to the right) and the y-axis denotes suction strength (increasing upwards). In the TEM images, the presence of vitreous ice can be observed as blurring at the edge of the lysozyme crystal. Looking at the MicroED diffraction patterns, high-resolution is observed by the presence of bright spots in the periphery of the diffraction pattern. It can be seen that the specimen prepared using high suction on a Quantifoil film having large hole size (TEM image (c) and MicroED diffraction pattern (d)) results in a dehydrated crystal, little vitreous ice, and a low ED resolution. On the other hand, the specimen prepared using low suction on a Quantifoil film having small hole size (TEM image (e) and MicroED diffraction pattern (f)) had too-thick vitreous ice and thus also had a poor ED resolution. The "ice rings" are clearly visible in the diffraction pattern. The specimens prepared using either high suction and small hole size (TEM image (a) and MicroED diffraction pattern (b)) or low suction and large hole size (TEM image (g) and MicroED diffraction pattern (h)) both provided satisfactory vitreous ice thickness and good ED resolution. Resolution of up to 1.6 A was obtained by specimen preparation using the inventive method, which is as good as the resolution obtainable using the normal (blotting) Vitrobot method.

Viscous samples

One of the most exciting aspects of the inventive method is that it can handle protein crystals which grow in very viscous mother liquid. The known deposit-blot-plunge method struggles to prepare specimens from samples having very viscous mother liquid. The latest developed methods such as inkjet-based methods are not either known to be able to prepare specimens from samples having very viscous mother liquid. In order to demonstrate the efficacy of the inventive method, we prepared specimens of a new protein sample, SaR2loxlll crystals, grown in very viscous mother liquid with 43% PEG400, using both the known blotting method and the inventive method. The resulting TEM images and diffraction patterns are shown in Figure 8. Images (a) to (c) illustrate the specimen prepared by Vitrobot using extreme conditions (multiple layers of filter paper on each side, large blotting force and long blotting time), whereas images (d) to (f) illustrate the specimen prepared using the inventive (horizontal) method. With the specimen prepared using the known Vitrobot method with heavy blotting, we can only observe a single hole on the TEM grid which can allow electrons to go through. In the area of this hole having the thinnest vitreous ice, we can find a crystal and obtain a diffraction pattern with low resolution (about 5 A). However, the exposure time we used to get one diffraction image is 20 s which is not feasible for solving the protein structure. Note also, that due to the low concentration of protein retained on the grid after blotting it took over 8 hours in order to find a suitable crystal for MicroED on the grid. However, using our horizontal method, we can get quite large areas with good ice thickness. Note for example that in images (d) and (e) the Quantifoil holes are observable. The resolution of electron diffraction pattern can reach to 3.2 A, and the exposure time for each diffraction image is 2 s. Finding a suitable crystal for diffraction on the specimen took approximately a couple of minutes. With this specimen preparation method, we can get very good 3D electron diffraction data. This is the first new protein crystal structure solved by MicroED that has very low sequence identity (36%) to any known proteins.

Continuous film grids

Specimens were prepared using Quantifoil continuous carbon grid R2/2, which has an ultrathin continuous layer of carbon applied on top of the holey carbon film. The specimens were prepared using the vertical method as described above, and the samples tested were apoferritin and GroEL. The resulting TEM images from GroEL specimens are shown in Figure 9, images a) to e). It was found that the continuous film resulted in a more homogenous ice thickness distribution across the grid. It is thought that this may be due to the continuous film providing a more even distribution of pressure across the entire grid.

Electron cryotomography

Electron cryotomograpy (cryoET) specimens can also be prepared by using the proposed method. We use yeast mitochondria as an example. The grid type used was Quantifoil R 3.5/1 with holes. Specimens were prepared using the "horizontal" method as described herein. The results are shown in Figure 10, images c) and d). For comparison, specimens were also prepared using the prior-art Vltrobot method with blotting. The results are shown in Figure 10, images a) and b).

It was found that in the specimen prepared by the proposed method, numerous mitochondria and gold beads were located in the middle of the holes, which is desirable for cryoET data collection.

In summary, the inventive method allows for cheap and simple specimen preparation with high retention of sample, and allows specimens to be prepared from samples in viscous mother liquid that would be difficult, if not impossible, to prepare by other means.

Claims

1. A method for cryo-electron microscopy (cryo-EM) specimen preparation, comprising the steps:

a) providing an EM sample grid;

b) applying a sample dispersed in a liquid onto at least a first face of the EM sample grid; c) providing a pressure gradient through the EM sample grid in order to force a portion of the liquid through the EM sample grid and remove the portion of liquid; and

d) immersing the EM sample grid into a cryogenic bath.

2. The method according to claim 1, wherein the step c) of providing a pressure gradient through the EM sample grid is performed by applying a suction to a second face of the EM sample grid, the second face being the opposite face to the first face.

3. The method according to claim 2, wherein in step c) the EM sample grid is clamped in an essentially vertical position by a tweezer, and wherein the suction is applied by a suction nozzle brought into in adjacency with the second face of the EM sample grid.

4. The method according to claim 3, wherein the suction nozzle is arranged to move in a scanning motion over the second face of the EM sample grid.

5. The method according to claim 2, wherein the suction is applied by a suction nozzle brought into contact with the second face of the EM sample grid, and further comprising a step of lifting and/or transferring the EM sample grid by using the suction applied by the suction nozzle.

6. The method according to claim 5, wherein an EM sample grid with applied sample is transferred to a position directly above the cryogenic bath using suction provided at the second face of the EM sample grid, and wherein suction is then removed, causing the EM sample grid to fall into the cryogenic bath and be frozen.

7. The method according to any one of claims 3-6, wherein the suction nozzle comprises a plurality of nozzle sub-orifices, each nozzle sub-orifice being arranged to provide an individual fluid flow direction and/or fluid flow rate through the nozzle sub-orifice.

8. The method according to claim 2, wherein in step c) the second face of the EM sample grid rests upon a porous support, and wherein the suction is applied through the porous support.

9. The method according to claim 1, wherein the step c) of providing a pressure gradient through the EM sample grid is performed by applying a positive pressure to the first face of the EM sample grid.

10. The method according to any one of the preceding claims, wherein the liquid is a viscous liquid, preferably an aqueous PEG solution or lipid cubic phase.

11. An auxiliary suction module for a cryo-EM specimen preparation apparatus, wherein the auxiliary suction module is adapted to provide a pressure gradient through an EM sample grid in order to force a portion of a liquid through the EM sample grid and remove the portion of liquid.

12. An auxiliary suction module according to claim 11, further adapted to apply a sample dispersed in a liquid onto at least a first face of the EM sample grid, and/or further adapted to immerse the EM sample grid into a cryogenic bath.

13. An auxiliary suction module for a cryo-EM specimen preparation apparatus, the auxiliary suction module comprising a mechanical arm, wherein the mechanical arm comprises a suction nozzle arranged to be connected to a source of partial vacuum, and wherein the mechanical arm is arranged to controllably provide translational motion of the suction nozzle, such that the suction nozzle may be brought into adjacency with a second face of an EM sample grid.

14. A cryo-EM specimen preparation apparatus, wherein the apparatus is adapted to provide a pressure gradient through an EM sample grid in order to force a portion of a liquid through the EM sample grid and remove the portion of liquid.

15. A cryo-EM specimen preparation apparatus according to claim 14, further adapted to apply a sample dispersed in a liquid onto at least a first face of the EM sample grid, and/or further adapted to immerse the EM sample grid into a cryogenic bath.

16. A cryo-EM specimen preparation apparatus, the apparatus comprising a retractable plunge rod and a bath volume arranged to accommodate a cryogenic bath, the retractable plunge rod being adapted to directly or indirectly hold an EM sample grid and to plunge the EM sample grid into the bath volume, characterised in that the apparatus comprises a mechanical arm, wherein the mechanical arm comprises a suction nozzle arranged to be connected to a source of partial vacuum, and wherein the mechanical arm is arranged to controllably provide translational motions of the suction nozzle, such that the suction nozzle may be brought into adjacency with a second face of an EM sample grid.