EMDB-8595: CryoEM Structure Refinement by Integrating NMR Chemical Shifts wi...

Basic information

• Entry: Database: EMDB / ID: 8595
• Title: CryoEM Structure Refinement by Integrating NMR Chemical Shifts with Molecular Dynamics Simulations
• Map data: HIV-1 CA hexamer

Sample

HIV-1 Capsid Protein Assembly

• Gag polyproteinGroup-specific antigen

• Function / homology: Retrovirus capsid, C-terminal / Zinc finger CCHC-type profile. / gag gene protein p24 (core nucleocapsid protein) / gag gene protein p17 (matrix protein) / Zinc knuckle / Zinc finger, CCHC-type superfamily / Gag protein p6 / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Retroviral matrix protein / Retrovirus capsid, N-terminal / Zinc finger, CCHC-type / Retroviral nucleocapsid protein Gag / Immunodeficiency lentiviral matrix, N-terminal / ISG15 antiviral mechanism / Gag protein p6 / viral budding via host ESCRT complex / host multivesicular body / viral nucleocapsid / host cell nucleus / host cell plasma membrane / virion membrane / structural molecule activity / RNA binding / zinc ion binding / Gag polyprotein
• Source: Human immunodeficiency virus type 1 (NEW YORK-5 ISOLATE) / virus
• Method: helical reconstruction / cryo EM / 5 Å resolution
• Authors: Zhao G / Zhang P
• Citation: Journal: J Phys Chem B / Year: 2017; Title: CryoEM Structure Refinement by Integrating NMR Chemical Shifts with Molecular Dynamics Simulations.; Authors: Juan R Perilla / Gongpu Zhao / Manman Lu / Jiying Ning / Guangjin Hou / In-Ja L Byeon / Angela M Gronenborn / Tatyana Polenova / Peijun Zhang; Abstract: Single particle cryoEM has emerged as a powerful method for structure determination of proteins and complexes, complementing X-ray crystallography and NMR spectroscopy. Yet, for many systems, the resolution of cryoEM density map has been limited to 4-6 Å, which only allows for resolving bulky amino acids side chains, thus hindering accurate model building from the density map. On the other hand, experimental chemical shifts (CS) from solution and solid state MAS NMR spectra provide atomic level data for each amino acid within a molecule or a complex; however, structure determination of large complexes and assemblies based on NMR data alone remains challenging. Here, we present a novel integrated strategy to combine the highly complementary experimental data from cryoEM and NMR computationally by molecular dynamics simulations to derive an atomistic model, which is not attainable by either approach alone. We use the HIV-1 capsid protein (CA) C-terminal domain as well as the large capsid assembly to demonstrate the feasibility of this approach, termed NMR CS-biased cryoEM structure refinement.
• Validation Report: PDB-ID: 5upw; ; SummaryFull reportAbout validation report
• Date: Deposition: Feb 4, 2017 / Header (metadata) release: Mar 1, 2017 / Map release: Mar 1, 2017 / Last update: Dec 6, 2017

Map

• File: emd_8595.map.gz (map file in CCP4 format, 379276 KB)
• Voxel size: X=Y=Z: 1.22 Å

Density

Contour Level:	0.035 (by author), 0.035 (movie #1):
Minimum - Maximum	-0.025930129 - 0.092899546
Average (Standard dev.)	3.2771706E-5 (0.0012119777)

Details

EMDB XML:

Space Group Number	1
Map Geometry	Axis order X Y Z Dimensions 456 456 456 Origin 0 0 0 Limit 455 455 455 Spacing 456 456 456
Cell	A=B=C: 556.32 Å α=β=γ: 90 deg.

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	1.22	1.22	1.22
M x/y/z	456	456	456
origin x/y/z	0.000	0.000	0.000
length x/y/z	556.320	556.320	556.320
α/β/γ	90.000	90.000	90.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	456	456	456
D min/max/mean	-0.026	0.093	0.000

Supplemental data

Sample components

Entire HIV-1 Capsid Protein Assembly

• Entire: Name: HIV-1 Capsid Protein Assembly / Number of components: 2

Component #1: protein, HIV-1 Capsid Protein Assembly

• Protein: Name: HIV-1 Capsid Protein Assembly / Recombinant expression: No
• Source: Species: Human immunodeficiency virus type 1 (NEW YORK-5 ISOLATE) / virus
• Source (engineered): Expression System: Escherichia coli / / bacteria /

Component #2: protein, Gag polyprotein

• Protein: Name: Gag polyproteinGroup-specific antigen / Recombinant expression: No
• Mass: Theoretical: 24.654268 kDa
• Source (engineered): Expression System: Human immunodeficiency virus type 1 (NEW YORK-5 ISOLATE) / virus

Experimental details

Sample preparation

• Specimen: Specimen state: helical array / Method: cryo EM
• Helical parameters: Axial symmetry: C1 (asymmetric) / Delta z: 6.94 Å / Delta phi: -31.13 deg.
• Sample solution: Specimen conc.: 2 mg/ml / pH: 8
• Vitrification: Instrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 295 K / Humidity: 100 %; Details: The assembled sample (1.5 microliter) was applied to the carbon side of a glow discharged perforated Quantifoil grid, followed by application of 3 microliter of low salt buffer (100 milimolar NaCl, 50 milimolar Tris pH 8.0) on the back side of the grid, and blotting, from the back side, with a filter paper, before plunge-freezing in liquid ethane

Electron microscopy imaging

• Experimental equipment: Model: Tecnai Polara / Image courtesy: FEI Company
• Imaging: Microscope: FEI POLARA 300
• Electron gun: Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 41 e/Ų / Illumination mode: FLOOD BEAM
• Lens: Magnification: 31000 X (nominal) / Cs: 2.2 mm / Imaging mode: BRIGHT FIELD / Defocus: 700 - 2200 nm
• Specimen Holder: Model: SIDE ENTRY, EUCENTRIC
• Camera: Detector: GATAN K2 (4k x 4k)

Image acquisition

• Image acquisition: Number of digital images: 523

Image processing

• Processing: Method: helical reconstruction
• 3D reconstruction: Algorithm: FOURIER SPACE / Software: RELION / Resolution: 5 Å / Resolution method: FSC 0.143 CUT-OFF

Atomic model buiding

Modeling #1

Refinement protocol: flexible / Refinement space: REAL
Input PDB model: 4XFX

4xfx

Output model

5upw