Methods for collecting electron diffraction patterns
Methods of collecting diffraction patterns from a microcrystal having an ordered array of a molecule are disclosed, which include using an exposure rate of at most 0.02 electrons per square angstrom per second on the microcrystal and using a direct electron detector to record electron diffraction patterns. Also disclosed are methods of determining a structural model for a molecule, identifying a material present in a trace amount within a sample, identifying a polymorph, and identifying the stereochemistry of a molecule.
1 . A method of collecting diffraction patterns from a microcrystal having an ordered array of a molecule, the method comprising:
subjecting the microcrystal having a first orientation or orientation group to a parallel electron beam for an exposure time less than 4 seconds in a transmission electron microscope (TEM) using an exposure rate of at most 0.02 electrons per square angstrom per second on the microcrystal;
recording at least one electron diffraction pattern (EDP) from the microcrystal having the first orientation or orientation group using a direct electron detector (DED); and
repeating said subjecting and said recording for additional orientations or orientation groups of the microcrystal to obtain an EDP set comprising said at least one EDP and additional EDPs.
2 . The method of claim 1 , wherein the microcrystal is rotated around an axis to attain each said orientation or orientation group.
3 . The method of claim 2 , wherein the microcrystal is rotated around the axis continuously.
4 . The method of claim 2 , wherein the EDP set is recorded as a movie in which each EDP is represented by a frame or by a function of multiple frames.
5 . The method of claim 4 , wherein the function is an average.
6 . The method of claim 2 , wherein the microcrystal is rotated around the axis by at least 20 degrees.
7 . The method of claim 1 , wherein the EDP set is obtained in less than 10 minutes.
8 . The method of claim 1 , wherein a total electron exposure on the microcrystal for each EDP in the EDP set is at most 1.2 electrons per square angstrom.
9 . The method of claim 1 , wherein said DED is used in an integration mode or in electron-counting mode.
10 . The method of claim 1 , wherein said molecule is a macromolecule.
11 . The method of claim 10 , wherein said macromolecule is a protein.
12 . The method of claim 1 , wherein the microcrystal has at least one dimension of at least 20 nm and at most 900 nm.
13 . The method of claim 1 , wherein said microcrystal comprises unit cells with an average volume of at least 10,000 cubic angstroms and at most 1,000,000 cubic angstroms.
14 . The method of claim 1 , wherein the method, as compared to a method that differs only in the use of an indirect complementary metal-oxide-semiconductor (CMOS) detector instead of a DED, reduces levels of radiation damage affecting the microcrystal, the detector, or a structural model obtained from the EDP set.
15 . The method of claim 1 , wherein the microcrystal is maintained under cryogenic conditions.
16 . The method of claim 15 , wherein the cryogenic conditions comprise liquid nitrogen.
17 . The method of claim 1 , wherein the TEM is operated at a voltage of at least 40 kilovolts and at most 400 kilovolts.
18 . The method of claim 17 , wherein the TEM is operated at a voltage of 200 kilovolts or 300 kilovolts.
19 . A method of determining a structural model for a molecule, comprising:
subjecting a microcrystal having a first orientation or orientation group to a parallel electron beam for an exposure time less than 4 seconds in a transmission electron microscope (TEM) using an exposure rate of at most 0.02 electrons per square angstrom per second on the microcrystal, wherein the microcrystal comprises an ordered array of the molecule;
recording at least one electron diffraction pattern (EDP) from the microcrystal having the first orientation or orientation group using a direct electron detector (DED);
repeating said subjecting and said recording for additional orientations or orientation groups of the microcrystal to obtain an EDP set comprising said at least one EDP and additional EDPs; and
determining a structural model for the molecule by processing the EDP set.
20 . A method of identifying a material present in a trace amount within a sample, the method comprising:
applying the sample to an electron microscopy (EM) grid;
subjecting the EM grid, at a plurality of orientations, to a parallel electron beam in a transmission electron microscope (TEM);
recording electron diffraction patterns (EDPs) from the EM grid at the plurality of orientations; and
identifying a material present in a trace amount within the sample by processing the EDPs, wherein the material comprises a molecule;
wherein the material comprises a polymorphic form of a microcrystal of the molecule and the microcrystal comprises unit cells with an average volume of at least 10,000 cubic angstroms and at most 1,000,000 cubic angstroms.
21 . A method of identifying the stereochemistry of a molecule, the method comprising:
obtaining a sample having a molecule with at least one known chiral center and at least one unknown chiral center;
applying the sample to an electron microscopy (EM) grid;
subjecting the EM grid, at a plurality of orientations, to a parallel electron beam in a transmission electron microscope (TEM);
recording electron diffraction patterns (EDPs) from the EM grid at the plurality of orientations; and
identifying the stereochemistry of the molecule based on the at least one known chiral center by processing the EDPs.
22 . The method of claim 21 , wherein said identifying the stereochemistry is based on a structural model determined from said processing, wherein the structural model has a resolution equal to or better than 3 angstroms.