[English] 日本語
Yorodumi
- EMDB-7540: Cryo-EM map of an HIV-1 reverse transcriptase initiation complex ... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-7540
TitleCryo-EM map of an HIV-1 reverse transcriptase initiation complex in magnesium chloride imaging buffer
Map dataHIV-1 RTIC core
Sample
  • Complex: HIV-1 reverse transcriptase initiation complex
    • Complex: HIV-1 reverse transcriptaseReverse transcriptase
    • Complex: tRNA lysine3 primer
    • Complex: HIV-1 RNA genome fragment
Biological speciesHuman immunodeficiency virus 1 / Human (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.2 Å
AuthorsLarsen KP / Chen DH / Puglisi JD / Puglisi EV
CitationJournal: Nature / Year: 2018
Title: Architecture of an HIV-1 reverse transcriptase initiation complex.
Authors: Kevin P Larsen / Yamuna Kalyani Mathiharan / Kalli Kappel / Aaron T Coey / Dong-Hua Chen / Daniel Barrero / Lauren Madigan / Joseph D Puglisi / Georgios Skiniotis / Elisabetta Viani Puglisi /
Abstract: Reverse transcription of the HIV-1 RNA genome into double-stranded DNA is a central step in viral infection and a common target of antiretroviral drugs . The reaction is catalysed by viral reverse ...Reverse transcription of the HIV-1 RNA genome into double-stranded DNA is a central step in viral infection and a common target of antiretroviral drugs . The reaction is catalysed by viral reverse transcriptase (RT) that is packaged in an infectious virion with two copies of viral genomic RNA each bound to host lysine 3 transfer RNA (tRNA), which acts as a primer for initiation of reverse transcription. Upon viral entry into cells, initiation is slow and non-processive compared to elongation. Despite extensive efforts, the structural basis of RT function during initiation has remained a mystery. Here we use cryo-electron microscopy to determine a three-dimensional structure of an HIV-1 RT initiation complex. In our structure, RT is in an inactive polymerase conformation with open fingers and thumb and with the nucleic acid primer-template complex shifted away from the active site. The primer binding site (PBS) helix formed between tRNA and HIV-1 RNA lies in the cleft of RT and is extended by additional pairing interactions. The 5' end of the tRNA refolds and stacks on the PBS to create a long helical structure, while the remaining viral RNA forms two helical stems positioned above the RT active site, with a linker that connects these helices to the RNase H region of the PBS. Our results illustrate how RNA structure in the initiation complex alters RT conformation to decrease activity, highlighting a potential target for drug action.
History
DepositionMar 9, 2018-
Header (metadata) releaseMay 2, 2018-
Map releaseMay 2, 2018-
UpdateMay 9, 2018-
Current statusMay 9, 2018Processing site: RCSB / Status: Released

-
Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.00703
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.00703
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7540.png

Downloads & links

-
Map

FileDownload / File: emd_7540.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHIV-1 RTIC core
Voxel sizeX=Y=Z: 1.286 Å
Density
Contour LevelBy AUTHOR: 0.00703 / Movie #1: 0.00703
Minimum - Maximum-0.006215456 - 0.03333984
Average (Standard dev.)-0.000013851448 (±0.0015925956)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 329.216 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.2861.2861.286
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z329.216329.216329.216
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0060.033-0.000

-
Supplemental data

-
Sample components

-
Entire : HIV-1 reverse transcriptase initiation complex

EntireName: HIV-1 reverse transcriptase initiation complex
Components
  • Complex: HIV-1 reverse transcriptase initiation complex
    • Complex: HIV-1 reverse transcriptaseReverse transcriptase
    • Complex: tRNA lysine3 primer
    • Complex: HIV-1 RNA genome fragment

-
Supramolecule #1: HIV-1 reverse transcriptase initiation complex

SupramoleculeName: HIV-1 reverse transcriptase initiation complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Details: Alternate buffer conditions containing magnesium chloride
Molecular weightTheoretical: 33 KDa

-
Supramolecule #2: HIV-1 reverse transcriptase

SupramoleculeName: HIV-1 reverse transcriptase / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Details: A cysteine mutation for crosslinking was introduced into helix H of p66 (Q258C). The protein used in this study also had the C280S mutation, introduced in prior structural work, and the ...Details: A cysteine mutation for crosslinking was introduced into helix H of p66 (Q258C). The protein used in this study also had the C280S mutation, introduced in prior structural work, and the E478Q mutation, introduced to eliminate RNase H activity.
Source (natural)Organism: Human immunodeficiency virus 1
Recombinant expressionOrganism: Escherichia coli (E. coli)

-
Supramolecule #3: tRNA lysine3 primer

SupramoleculeName: tRNA lysine3 primer / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Details: Chemically synthesized and extended tRNA lysine3 primer. Modified nucleotide containing a N2-cystamine was placed at position 71. The tRNA primer has been extended by one ddCTP, bringing its ...Details: Chemically synthesized and extended tRNA lysine3 primer. Modified nucleotide containing a N2-cystamine was placed at position 71. The tRNA primer has been extended by one ddCTP, bringing its total length in the full complex to 77 nucleotides.
Source (natural)Organism: Human (human)

-
Supramolecule #4: HIV-1 RNA genome fragment

SupramoleculeName: HIV-1 RNA genome fragment / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #4
Details: HIV-1 RNA genome fragment of 101 nucleotides in length. Contains the primer binding site (PBS), primer activation signal (PAS), A-rich loop, and C-rich region. Generated via T7 transcription and PAGE purified.
Source (natural)Organism: Human immunodeficiency virus 1

-
Experimental details

-
Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

-
Sample preparation

Concentration4.5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
75.0 mMNaClSodium chloridesodium chloride
2.5 mMMgCl2magnesium chloride
10.0 mMC4H11NO3Tris-HClTris
0.25 % w/vbeta-octyl glucoside

Details: Beta-OG was added just prior to freezing.
GridMaterial: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / Chamber temperature: 292 K / Instrument: LEICA EM GP
Details: Blotted for 2.8 sec before plunging into liquid ethane..
DetailsSample was monodisperse.

-
Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 38880 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.26 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 29000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 2-60 / Number real images: 898 / Average exposure time: 12.0 sec. / Average electron dose: 75.0 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

-
Image processing

Particle selectionNumber selected: 148523
CTF correctionSoftware - Name: Gctf
Startup modelType of model: INSILICO MODEL
In silico model: An initial 3D model was obtained using EMAN2 based on initial selected 2D classes.
Initial angle assignmentType: NOT APPLICABLE / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION
Final reconstructionNumber classes used: 1 / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 8.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 67346

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more