Cryo-EM validation

tools in CCP-EM

Martyn Winn

20 November 2020

CCP-EM & CCP4 | RCaH

eBIC | DLS

Collaborative Computational Project for Electron cryo-Microscopy

Core team hosted by Research Complex at Harwell, alongside CCP4 team.

Wide network of collaborators.

What is CCP-EM?

Tom

Burnley

Colin

Palmer

Agnel

Joseph

Jola

Mirecka

Matt

Iadanza

Build UK cryo-EM community• Annual conference: CCP-EM Spring Symposium• Talks recorded, available on YouTube

Software training workshopsMailing listWorkshops for developersBenchmarking

CCP-EM software suite• Data processing tools for cryo-EM• Free for academic use, charge for industrial

Support for UK national facility at eBIC

Highlights

Replace job and metadata handling with new modular, flexible

Python layer. Unified approach for RELION and CCP-EM suite.

Sjors

Scheres

Standards / validation

Training

schools

Machine learning for segmentation of molecular maps

Support

for eBIC

Integrates tools for EM data processing

Collection of programs with common look-and-feel

Download from ccpem.ac.uk (Linux & Mac)

Stable v1.5. Use nightly for latest updates

Bugs & requests: ccpem@stfc.ac.uk

Jobs run via:

Task GUI windows

Python API

Individual program executables

CCP-EM software suite

> ccpem main GUI

> ccpem-mrc-to-mtz task GUI

> ccpem-python scripting

> refmac5 executables

Validation overview

Of course, validation should be part of the structure determination process!

CCP-EM GUI allows sanity checking of input and interpretation of output.

Some tasks are more focussed on validation.

Designed for off-line validation during structure determination, prior to deposition.Complementary to EMDB / PDB.

Confidence maps

Carsten’s talk yesterday

Is there map support for atomic models?Model validation.

CryoEFNaydenova, K and Russo, CJ "Measuring the effects of particle

orientation to improve the efficiency of electron cryomicroscopy" Nature

Communications, 8, Article number: 629 (2017).

e.g. run_data.starfrom Relion Refine3D

V1.1 in CCP-EM. Quantify spread of particle angle distribution, recommends tilt angles to minimize bias

TEMPy: DiffMap

Map-Map or Map-ModelMaps are scaled based on average resolution dependent amplitude fall-offs of the maps

ADP-AlFxbound to kinesin-

6 motor domain

W1

4N68

K11

L83

L80H72

EMD-3622 (4.4Å) vs EMD-3621 (6.1 Å)

EMD-3488 (3.2Å) vs 5NI1_mut

Global or local scaling.

Latter can minimize effect of variable local resolution

Joseph et al. JCIM (2020)

Hemoglobinrotamer errors

EMDADeveloped by Rangana, see following talk.

Importable Python3 library for EM map and model manipulations:

Resolution related functionalities Map statistics calculation in Image and Fourier space Local correlation map calculation for map and model validation FSC based map-model validation

Included in CCPEM v1.5

No task, command line only (for now).In terminal window:emda -h

emda fsc --map1 foo_half_map_1.map

--map2 foo_half_map_2.map

ProSHADESymmetry/pseudo-symmetry detection

TEMPy-LocScore

Segment based Manders’ Overlap Coefficient (SMOC)

An overlap coefficient is calculated over

voxels covered by each residue (and the

local neighborhood)

Neighbourhood in sequence or space.

Joseph et al. 2016 , Farabella et al. 2015,

TEMPy: Scores

Difference

from best

Joseph et al. JSB, 2017

Several global scores (volume / surface / overlap).Compare alternative atomic models.

Refmac: Half-map cross-validation

Brown et al. 2015

Reciprocal space model refinement.CCP-EM task includes half-map validation.Test for overfitting of model to map.

weight matrix = 0.001

weight matrix = 0.1

Increase weight on fit to map

Validation: model taskDedicated validation task with several metricsAgnel’s talk

Geometry (bond/angle/dihedral): MolprobityCA geometry / peptides: (CaBLAM)

Bfactor distribution

Local fit in density: (TEMPy SMOC)Model map FSC / FSCavg: (Refmac)

Secondary structure prediction (Jpred)

Uses local programs (except last)Willams et a. 2018, Chen et al. 2015, Brown et al. 2015,

Joseph et al. 2016

Atomic B-factor distributionFrom input model – no refinement performed.

CCPEM - Model validation

Local fit to map: SMOC

Fixing in Coot

Launches Coot with map, model and to-do list

Future directions

Machine learning

Automated annotation, unusual features

Haruspex (in alpha mode). Deep learning approach to identify secondary structures in maps. Thorn A et al. Angewandte (2020)

Deposition

Gemmi project.Data harvesting.Collation of deposition files / metadata.Validation reports.

Integration with Relion

Improve data / metadata flow.Make intermediate steps available for validation.

CCP-EM core team

● Tom Burnley

● Colin Palmer

● Agnel Praveen Joseph

● Jola Mirecka

● Matt Iadanza

CCP4 core team

STFC SCD

● Alan Kyffin

DLS / eBIC staff

Birkbeck

● Maya Topf & group

University of Manchester

● Alan Roseman & group

AcknowledgementsImperial College London

● Chris Aylett

Francis Crick Institute

● Peter Rosenthal

University of Leeds

● Neil Ranson

● Becky Thompson

EBI

● Gerard Kleywegt

● Ardan Patwardhan

● Zhe Wang

MRC-LMB

● Garib Murshudov

● Sjors Scheres

● Paul Emsley

● Rob Nicholls

● Rangana Warshamanage

● Katerina Naydenova

University of York

● Kevin Cowtan

● Soon Wen ‘Scott’ Hoh

● Jon Agirre

TU Delft

● Arjen Jakobi

EMBL / FZ Jülich

● Max Beckers

● Carsten Sachse

University of Hamburg

● Andrea Thorn

… and others!