currently used for defining what is an atomic- or high-resolution structure. While it
is very common to use the term “atomic resolution” for cryo-EM resolutions better
than 3.5 Å, the crystallography definition of the term “atomic resolution” means
the resolution is 1.2 Å or better [1] and ultra-high resolution means 0.95 Å or
better ([83] and references cited therein). Similarly, 1.8 Å or better is called high
resolution [84], 3.0 Å or better up to 1.9 Å is treated as medium resolution while
low resolution is between 4 and 3.1 Å. Resolution below 4 Å is considered as poor
resolution in protein crystallography. While the method of estimation of resolution
is quite different between crystallography and cryo-EM techniques, the conventions
for using the terms should be consistent, irrespective of the method. Hence, the
author would like to suggest that it is necessary for the cryo-EM field to maintain
consistency in the future, while using the terms ultra-high, atomic, high, medium,
and low resolution.
3.2 Model Building
If the resolution of the 3D reconstruction (i.e., the electron potential map) is sufficiently high, e.g., better than 3 or 4 Å, it is often possible to build ab initio atomic
model and do refinement with the EM map using known chemical constraints/
restraints. If X-ray crystallography coordinates of the segment or its homologues
are available, one can rigid body fit the segment coordinates into the cryo-EM map
using programs like UCSF Chimera [85]. Where the resolution of the 3D reconstruction map is limited to worse than 4 Å, combining crystallography and
cryo-EM as a hybrid method is a powerful tool to obtain a pseudo-atomic model
(s). Iterative rounds of model building using programs like Coot [86], O [87] and
refinement using programs like Coot, refmac [88], or PHENIX
real-space-refinement [89] are carried out. De novo backbone tracing and model
building can be carried out using programs like Pathwalking and Gorgon [90]; it
can also build macromolecular assemblies at non-atomic resolution [90]. When the
cryo-EM map shows variation by domain movements or flexibility to the available
protein coordinates, programs like FlexEM [91, 92] and MDFF [93] with its
graphical user interface VMD [94] can be used to flexibly fit the coordinates in the
EM map. The fitted model and EM map can be visualized in programs like PyMOL
[95]/Chimera [85] to generate publication quality figures.
3.3 Validation
Validation in cryo-EM reconstruction is important to avoid errors in particle
alignment, reference bias, over-fitting of atomic coordinates, and over-estimation of
resolution. Validation tools for cryo-EM similar to the free R value (R free ) in X-ray
crystallography [96] have been introduced in 2003 by Joachim and co-workers [97]
390
R. Natesh
is very common to use the term “atomic resolution” for cryo-EM resolutions better
than 3.5 Å, the crystallography definition of the term “atomic resolution” means
the resolution is 1.2 Å or better [1] and ultra-high resolution means 0.95 Å or
better ([83] and references cited therein). Similarly, 1.8 Å or better is called high
resolution [84], 3.0 Å or better up to 1.9 Å is treated as medium resolution while
low resolution is between 4 and 3.1 Å. Resolution below 4 Å is considered as poor
resolution in protein crystallography. While the method of estimation of resolution
is quite different between crystallography and cryo-EM techniques, the conventions
for using the terms should be consistent, irrespective of the method. Hence, the
author would like to suggest that it is necessary for the cryo-EM field to maintain
consistency in the future, while using the terms ultra-high, atomic, high, medium,
and low resolution.
3.2 Model Building
If the resolution of the 3D reconstruction (i.e., the electron potential map) is sufficiently high, e.g., better than 3 or 4 Å, it is often possible to build ab initio atomic
model and do refinement with the EM map using known chemical constraints/
restraints. If X-ray crystallography coordinates of the segment or its homologues
are available, one can rigid body fit the segment coordinates into the cryo-EM map
using programs like UCSF Chimera [85]. Where the resolution of the 3D reconstruction map is limited to worse than 4 Å, combining crystallography and
cryo-EM as a hybrid method is a powerful tool to obtain a pseudo-atomic model
(s). Iterative rounds of model building using programs like Coot [86], O [87] and
refinement using programs like Coot, refmac [88], or PHENIX
real-space-refinement [89] are carried out. De novo backbone tracing and model
building can be carried out using programs like Pathwalking and Gorgon [90]; it
can also build macromolecular assemblies at non-atomic resolution [90]. When the
cryo-EM map shows variation by domain movements or flexibility to the available
protein coordinates, programs like FlexEM [91, 92] and MDFF [93] with its
graphical user interface VMD [94] can be used to flexibly fit the coordinates in the
EM map. The fitted model and EM map can be visualized in programs like PyMOL
[95]/Chimera [85] to generate publication quality figures.
3.3 Validation
Validation in cryo-EM reconstruction is important to avoid errors in particle
alignment, reference bias, over-fitting of atomic coordinates, and over-estimation of
resolution. Validation tools for cryo-EM similar to the free R value (R free ) in X-ray
crystallography [96] have been introduced in 2003 by Joachim and co-workers [97]
390
R. Natesh