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Case study: Using cryoSPARC to process the Plasmodium falciparum 80S ribosome

Refine the EMPIAR-10028 ribosome structure to 3.2 ‎Å from 105k particles, in only 40 minutes on a single GPU, using cryoSPARC's new optimized code.
*Scale bar: 25 ‎Å. Showing PDB3J79 and PDB3J7A docked into a cryoSPARC density map.*

This post is a walkthrough of processing the 80S Ribosome data from EMPIAR-10028 in cryoSPARC, including both ab-initio structure determination (taking approximately 50 minutes) and high-resolution gold-standard refinement to 3.2 ‎Å (taking approximately 40 minutes). All experiments were run on a single GPU workstation with one NVIDIA Titan X (M), an Intel i7 6-core CPU, 64 GB CPU RAM, and a 512 GB local SSD.
Note: The results described here use the new optimized code incorporated in cryoSPARC v0.2.47. In earlier versions of cryoSPARC, refinement took approximately 2.2 hours.

Ab-initio reconstruction in cryoSPARC

cryoSPARC allows for rapid, unbiased single- and multi-class reconstructions from both homogeneous and heterogeneous datasets, without any initial references or prior structural knowledge. Ab-initio reconstruction minimizes the risk of bias from an initial reference, and when used to perform heterogeneous reconstruction, can be used to discover multiple states or structures present in a dataset, directly from the data itself.

We performed a single-class ab-initio reconstruction on the Plasmodium falciparum 80S ribosome dataset (EMPIAR-10028).

Single-class ab-initio reconstruction of the 80S ribosome

Once a dataset of particles and CTF information has been loaded into cryoSPARC, it can simply be selected from the list:

selected 80S ribosome dataset

We started a new experiment relating to the 80S ribosome dataset by navigating to the Experiments page and clicking 'New Experiment'. The drop down menu displays the experiments applicable/available to/for the chosen dataset in purple:

available experiments

When starting an experiment in cryoSPARC, structure and particle set inputs are selected directly from the experiments page. For ab-initio reconstruction, no initial structures need to be selected, but the number of ab-initio classes to attempt reconstruction for must be specified. The default number of ab-initio classes is set to 1. The value need not be changed if, as in this case, you are performing a homogeneous (i.e., single class) ab-initio reconstruction. For heterogeneous (i.e., multiple class) reconstruction, you have the option to input the desired number of ab-initio classes:

option to modify the number of classes

Click the purple 'Ab-initio' button to set up the experiment. CryoSPARC provides default parameters for each task type, and no modifications are necessary to achieve the reported results. The experiment name can also be changed by clicking on it and typing the new name:

default parameters

Simply click 'Launch' and 'Enqueue' to commence the experiment and view/download plots and results in real-time. For example, three axis-aligned slices of each ab-initio structure are created in the results stream that can be useful for inspecting the progress of reconstruction:

slice plots

Several other useful plots are provided in-line as well. We downloaded the below viewing direction plot in PNG format directly from the results stream. PDF and raw data formats are also available for making publication figures. The below plot shows a typical viewing direction distribution over azimuth and elevation angles for this dataset:

viewing direction

CryoSPARC jobs will continue to run even if you close your browser.
When a job is completed, the indicator on your browser tab changes from purple (experiment running) to green (and is also indicated similarly on the Experiments page):


Reconstruction results

The full ab-initio job took 48 minutes. The result structures and other meta data (alignment estimates, class assignments, etc) can be downloaded directly in your browser from the result stream by clicking on the Download File link.

download result

40 min gold-standard refinement to 3.2 ‎Å

Once an ab-initio structure has been determined in cryoSPARC, it can be directly refined to high-resolution using gold-standard dataset splitting and FSC calculation, corrected for masking via noise substitution. Generally, ab-initio reconstruction can resolve features out to a resolution of approximately 8 Å.

We refined the results of the reconstruction above to obtain a 3.2 ‎Å structure of the ribosome, equal to that achieved in the published result in eLife. This was completed, starting from a low-pass filtered version of the ab-initio result, in 40 minutes.

From the 'Experiments' page, we can view the result of the completed ab-initio run. The orange flag displays the proportion of all particles in the experiment that belong to a particular class - here, as there was only a single class, only one structure is displayed and 100% of the particles belong to that class. Following a heterogeneous ab-initio run, multiple structures would be displayed corresponding with the number of classes that were requested:

view result

To start a refinement of the structure produced during the ab-initio run, simply click 'Structure' below the experiment card. This selects the single structure on which we want to perform the refinement:

new from complete

Note 1: It is also possible to include the particles corresponding to a particular class if you want to include them in the refinement; however, this is not required for a single-class refinement since we want to use all particles in the dataset.
Note 2: Each structure result can be selected multiple times, for instance if the goal is to initiate a multi-class reconstruction with several classes starting from a single reference structure.

Next, click 'New Experiment' to display the experiments that can be performed on the selection - this time, Refinement is available and we select that from the list:

new refinement

As is the case with ab-initio reconstruction in cryoSPARC, all of the parameters in a refinement experiment have a default value and none need to be changed, except if you wish to enforce symmetry during refinement. In this case we can leave C1 symmetry as is:

enforce symmetry

Other parameters that may be useful to experiment with include dynamic masking thresholds and the box size of the refinement result.
Click 'Launch' and 'Enqueue' to commence the experiment and view/download plots and results in real-time.

Refinement results

Once started, the cryoSPARC refinement will proceed through iterations of alignment and reconstruction on independent half-sets of the data. Convergence is assessed by measuring the change in resolution over iterations. After a few iterations, cryoSPARC refinement for the 80S ribosome map completes. Three axis-aligned slices are shown in the results stream:

The dynamic refinement mask automatically generated by cryoSPARC is also shown, in three axis-aligned slices. The mask is generated by thresholding the density and expanding the resulting mask with a minimum distance and soft cosine edge.

The gold-standard FSC plot is also provided, revealing a 3.2 ‎Å result at the FSC=0.143 level.

CryoSPARC is developed and supported by Structura Biotechnology Inc. To request beta access to cryoSPARC, please visit cryosparc.com. Already using cryoSPARC? Find out more in the Guide and Discussion Forum.

For commercial use, please contact us at sales@structura.bio.