81. Kucukelbir A, Sigworth FJ, Tagare HD (2014) Quantifying the local resolution of cryo-EM
density maps. Nat Methods 11:63–65
82. Cardone G, Heymann JB, Steven AC (2013) One number does not fit all: mapping local
variations in resolution in cryo-EM reconstructions. J Struct Biol 184:226–236
83. Natesh R, Manikandan K, Bhanumoorthy P, Viswamitra MA, Ramakumar S (2003)
Thermostable xylanase from Thermoascus aurantiacus at ultrahigh resolution (0.89 A) at
100 K and atomic resolution (1.11 A) at 293 K refined anisotropically to small-molecule
accuracy. Acta Crystallogr D Biol Crystallogr 59:105–117
84. Natesh R, Bhanumoorthy P, Vithayathil PJ, Sekar K, Ramakumar S, Viswamitra MA (1999)
Crystal structure at 1.8 A resolution and proposed amino acid sequence of a thermostable
xylanase from Thermoascus aurantiacus. J Mol Biol 288:999–1012
85. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE
(2004) UCSF Chimera–a visualization system for exploratory research and analysis.
J Comput Chem 25:1605–1612
86. Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta
Crystallogr D Biol Crystallogr 60:2126–2132
87. Jones TA (2004) Interactive electron-density map interpretation: from INTER to O. Acta
Crystallogr D Biol Crystallogr 60:2115–2125
88. Brown A, Long F, Nicholls RA, Toots J, Emsley P, Murshudov G (2016) Tools for
macromolecular model building and refinement into electron cryo-microscopy reconstructions. Acta Crystallogr D Biol Crystallogr 71:136–153
89. Echols N, Moriarty NW, Klei HE, Afonine PV, Bunkoczi G, Headd JJ, McCoy AJ,
Oeffner RD, Read RJ, Terwilliger TC, Adams PD (2014) Automating crystallographic
structure solution and refinement of protein-ligand complexes. Acta Crystallogr D Biol
Crystallogr 70:144–154
90. Baker ML, Baker MR, Hryc CF, Ju T, Chiu W (2012) Gorgon and pathwalking:
macromolecular modeling tools for subnanometer resolution density maps. Biopolymers
97:655–668
91. Topf M, Lasker K, Webb B, Wolfson H, Chiu W, Sali A (2008) Protein structure fitting and
refinement guided by cryo-EM density. Structure 16:295–307
92. Joseph AP, Malhotra S, Burnley T, Wood C, Clare DK, Winn M, Topf M (2016) Refinement
of atomic models in high resolution EM reconstructions using Flex-EM and local
assessment. Methods 100:42–49
93. Trabuco LG, Villa E, Schreiner E, Harrison CB, Schulten K (2009) Molecular dynamics
flexible fitting: a practical guide to combine cryo-electron microscopy and X-ray
crystallography. Methods 49:174–180
94. Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. J Mol
Graph 14(33–8):27–28
95. Schrodinger LLC (2015) The PyMOL molecular graphics system, Version 1.8 in
96. Brunger AT (1992) Free R value: a novel statistical quantity for assessing the accuracy of
crystal structures. Nature 355:472–475
97. Shaikh TR, Hegerl R, Frank J (2003) An approach to examining model dependence in EM
reconstructions using cross-validation. J Struct Biol 142:301–310
98. Chen S, McMullan G, Faruqi AR, Murshudov GN, Short JM, Scheres SH, Henderson R
(2013) High-resolution noise substitution to measure overfitting and validate resolution in
3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy
135:24–35
99. Rosenthal PB, Rubinstein JL (2015) Validating maps from single particle electron
cryomicroscopy. Curr Opin Struct Biol 34:135–144
100. Scheres SH, Chen S (2012) Prevention of overfitting in cryo-EM structure determination.
Nat Methods 9:853–854
101. Murray SC, Flanagan J, Popova OB, Chiu W, Ludtke SJ, Serysheva II (2013) Validation of
cryo-EM structure of IP(3)R1 channel. Structure 21:900–909
Single-Particle cryo-EM as a Pipeline for Obtaining Atomic …
399
density maps. Nat Methods 11:63–65
82. Cardone G, Heymann JB, Steven AC (2013) One number does not fit all: mapping local
variations in resolution in cryo-EM reconstructions. J Struct Biol 184:226–236
83. Natesh R, Manikandan K, Bhanumoorthy P, Viswamitra MA, Ramakumar S (2003)
Thermostable xylanase from Thermoascus aurantiacus at ultrahigh resolution (0.89 A) at
100 K and atomic resolution (1.11 A) at 293 K refined anisotropically to small-molecule
accuracy. Acta Crystallogr D Biol Crystallogr 59:105–117
84. Natesh R, Bhanumoorthy P, Vithayathil PJ, Sekar K, Ramakumar S, Viswamitra MA (1999)
Crystal structure at 1.8 A resolution and proposed amino acid sequence of a thermostable
xylanase from Thermoascus aurantiacus. J Mol Biol 288:999–1012
85. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE
(2004) UCSF Chimera–a visualization system for exploratory research and analysis.
J Comput Chem 25:1605–1612
86. Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta
Crystallogr D Biol Crystallogr 60:2126–2132
87. Jones TA (2004) Interactive electron-density map interpretation: from INTER to O. Acta
Crystallogr D Biol Crystallogr 60:2115–2125
88. Brown A, Long F, Nicholls RA, Toots J, Emsley P, Murshudov G (2016) Tools for
macromolecular model building and refinement into electron cryo-microscopy reconstructions. Acta Crystallogr D Biol Crystallogr 71:136–153
89. Echols N, Moriarty NW, Klei HE, Afonine PV, Bunkoczi G, Headd JJ, McCoy AJ,
Oeffner RD, Read RJ, Terwilliger TC, Adams PD (2014) Automating crystallographic
structure solution and refinement of protein-ligand complexes. Acta Crystallogr D Biol
Crystallogr 70:144–154
90. Baker ML, Baker MR, Hryc CF, Ju T, Chiu W (2012) Gorgon and pathwalking:
macromolecular modeling tools for subnanometer resolution density maps. Biopolymers
97:655–668
91. Topf M, Lasker K, Webb B, Wolfson H, Chiu W, Sali A (2008) Protein structure fitting and
refinement guided by cryo-EM density. Structure 16:295–307
92. Joseph AP, Malhotra S, Burnley T, Wood C, Clare DK, Winn M, Topf M (2016) Refinement
of atomic models in high resolution EM reconstructions using Flex-EM and local
assessment. Methods 100:42–49
93. Trabuco LG, Villa E, Schreiner E, Harrison CB, Schulten K (2009) Molecular dynamics
flexible fitting: a practical guide to combine cryo-electron microscopy and X-ray
crystallography. Methods 49:174–180
94. Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. J Mol
Graph 14(33–8):27–28
95. Schrodinger LLC (2015) The PyMOL molecular graphics system, Version 1.8 in
96. Brunger AT (1992) Free R value: a novel statistical quantity for assessing the accuracy of
crystal structures. Nature 355:472–475
97. Shaikh TR, Hegerl R, Frank J (2003) An approach to examining model dependence in EM
reconstructions using cross-validation. J Struct Biol 142:301–310
98. Chen S, McMullan G, Faruqi AR, Murshudov GN, Short JM, Scheres SH, Henderson R
(2013) High-resolution noise substitution to measure overfitting and validate resolution in
3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy
135:24–35
99. Rosenthal PB, Rubinstein JL (2015) Validating maps from single particle electron
cryomicroscopy. Curr Opin Struct Biol 34:135–144
100. Scheres SH, Chen S (2012) Prevention of overfitting in cryo-EM structure determination.
Nat Methods 9:853–854
101. Murray SC, Flanagan J, Popova OB, Chiu W, Ludtke SJ, Serysheva II (2013) Validation of
cryo-EM structure of IP(3)R1 channel. Structure 21:900–909
Single-Particle cryo-EM as a Pipeline for Obtaining Atomic …
399