EMAN2: An extensible image processing suite for electron microscopy

Tang, Guang Yi; Peng, Liwei; Baldwin, Philip R.; Mann, Deepinder S.; Jiang, Wen; Rees, Ian; Ludtke, Steven J.

doi:10.1016/j.jsb.2006.05.009

Cited by 2,867 publications

(2,652 citation statements)

References 33 publications

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“…2f, 3f, 6f, 7f) by shifting their coordinates 0.5 Å with shake in PHENIX and building their corresponding densities in Chimera 42 from the shaken models. FSC was calculated between the densities from the shaken models, the half maps used in PHENIX refinement (work), the second half-maps (free) and the unfiltered sum maps, using EMAN2 45 . The local resolutions in the transmembrane regions of our hTRPV6-nanodiscs and hTRPV6-R470E maps reached 2.5 Å as estimated by ResMap 41 .…”

Section: Methodsmentioning

confidence: 99%

Opening of the human epithelial calcium channel TRPV6

McGoldrick

Singh

Saotome

et al. 2017

Nature

189

322

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Ca2+-selective transient receptor potential vanilloid subfamily member 6 (TRPV6) channels play a critical role in calcium uptake in epithelial tissues1–4. Altered TRPV6 expression is associated with a variety of human diseases5, including cancers6. TRPV6 channels are constitutively active1,7,8 and their open probability depends on the lipidic composition of the membrane, increasing significantly in the presence of phosphatidylinositol 4,5-bisphosphate (PIP2)7,9. We previously solved crystal structures of detergent-solubilized rat TRPV6 in the closed state10,11. Corroborating previous electrophysiological studies3, these structures demonstrated that the Ca2+ selectivity of TRPV6 arises from a ring of aspartate side chains in the selectivity filter that tightly binds Ca2+. However, it has remained unknown how TRPV6 channels open and close their pores for ion permeation. Here we present cryo-EM structures of human TRPV6 in the open and closed states. The channel selectivity filter adopts similar conformations in both states, consistent with its explicit role in ion permeation. The iris-like channel opening is accompanied by an α-to-π helical transition in the pore-lining S6 helices at an alanine hinge just below the selectivity filter. As a result of this transition, the S6 helices bend and rotate, exposing different residues to the ion channel pore in the open and closed states. This novel gating mechanism, which defines the constitutive activity of TRPV6, is unique for tetrameric ion channels and provides new structural insights for understanding their diverse roles in physiology and disease.

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Section: Methodsmentioning

confidence: 99%

Opening of the human epithelial calcium channel TRPV6

McGoldrick

Singh

Saotome

et al. 2017

Nature

189

322

View full text Add to dashboard Cite

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“…The negative stain datasets were processed using EMAN2 and RELION‐1.3 (Tang et al , 2007; Scheres, 2012). A total of 25,669 and 19,607 particles were semi‐automatically picked using e2boxer.py from the EMAN2 package from Pol I PIC (+DNA) and Pol I PIC (−DNA), respectively.…”

Section: Methodsmentioning

confidence: 99%

Structural insights into transcription initiation by yeast RNA polymerase I

et al. 2017

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In eukaryotic cells, RNA polymerase I (Pol I) synthesizes precursor ribosomal RNA (pre‐rRNA) that is subsequently processed into mature rRNA. To initiate transcription, Pol I requires the assembly of a multi‐subunit pre‐initiation complex (PIC) at the ribosomal RNA promoter. In yeast, the minimal PIC includes Pol I, the transcription factor Rrn3, and Core Factor (CF) composed of subunits Rrn6, Rrn7, and Rrn11. Here, we present the cryo‐EM structure of the 18‐subunit yeast Pol I PIC bound to a transcription scaffold. The cryo‐EM map reveals an unexpected arrangement of the DNA and CF subunits relative to Pol I. The upstream DNA is positioned differently than in any previous structures of the Pol II PIC. Furthermore, the TFIIB‐related subunit Rrn7 also occupies a different location compared to the Pol II PIC although it uses similar interfaces as TFIIB to contact DNA. Our results show that although general features of eukaryotic transcription initiation are conserved, Pol I and Pol II use them differently in their respective transcription initiation complexes.

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“…Images were recorded using the direct electron detector DE20 (1.54 Å per pixel) using a total dose of 30 electrons/Å 2 . Frames from 2 to 29 were aligned using EMAN2 (Tang et al , 2007) and IMOD (Kremer et al , 1996) software. The CTF was estimated using CTFFIND3 (Mindell & Grigorieff, 2003) and correction was done by phase flipping using BSOFT (Heymann, 2001) on the aligned frames.…”

Section: Methodsmentioning

confidence: 99%

Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery

et al. 2017

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Type IV secretion (T4S) systems are versatile bacterial secretion systems mediating transport of protein and/or DNA. T4S systems are generally composed of 11 VirB proteins and 1 VirD protein (VirD4). The VirB1‐11 proteins assemble to form a secretion machinery and a pilus while the VirD4 protein is responsible for substrate recruitment. The structure of VirD4 in isolation is known; however, its structure bound to the VirB1‐11 apparatus has not been determined. Here, we purify a T4S system with VirD4 bound, define the biochemical requirements for complex formation and describe the protein–protein interaction network in which VirD4 is involved. We also solve the structure of this complex by negative stain electron microscopy, demonstrating that two copies of VirD4 dimers locate on both sides of the apparatus, in between the VirB4 ATPases. Given the central role of VirD4 in type IV secretion, our study provides mechanistic insights on a process that mediates the dangerous spread of antibiotic resistance genes among bacterial populations.

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EMAN2: An extensible image processing suite for electron microscopy

Cited by 2,867 publications

References 33 publications

Opening of the human epithelial calcium channel TRPV6

Opening of the human epithelial calcium channel TRPV6

Structural insights into transcription initiation by yeast RNA polymerase I

Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery

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